Bacterial Identification Methods
1. Acetamide Utilization 2. Acetate Utilization 3. l-Alanine-7-amido-4-methylcourmar
• Purpose: Differentiate organisms based on the ability to use • Purpose: Differentiate organisms based on the ability to use • Purpose: Distinguish aerobic g
acetamide as the sole carbon source. acetate as the sole carbon source. coccobacilli that may appear g
• Principle: • Principle: • Principle:
o Bacteria producing acylamidase deaminate o Utilization of sodium acetate leads to alkaline pH, o Detects aminopeptida
acetamide, releasing ammonia.
turning the medium blue. negative organisms pr
o Ammonia increases pH, changing the medium from light.
green to royal blue. • Expected Results:
• Expected Results:
• Expected Results: o Positive: Blue color due to alkaline reaction.
o Positive: Blue fluoresc
o Positive: Blue color indicates acetamide utilization. o Negative: No color change; medium remains green.
o Negative: No fluoresc
o Negative: No color change; medium remains green. • Limitations:
• Limitations:
• Limitations: o Some E. coli strains may give false negatives.
o May not work with ob
o Heavy inoculum may lead to false negatives. • Quality Control:
• Quality Control:
• Quality Control: o Positive: Escherichia coli (growth; blue).
o Positive: Escherichia c
o Positive: Pseudomonas aeruginosa (growth; blue o Negative: Shigella sonnei (little growth; green).
color). o Negative: Staphylococ
o Negative: Escherichia coli (no growth; green color).
Source: Bailey’s and Scotts Diagnostic Microbiology (15th Edition)
, 4. Bacitracin Susceptibility Test 5. Bile Esculin Test 6. Bile Solubility Test
• Purpose: Differentiate β-hemolytic group A streptococci • Purpose: Identify enterococci and group D streptococci by • Purpose: Differentiate Streptoc
(susceptible) from other streptococci and distinguish their ability to hydrolyze esculin in the presence of bile. from other alpha-hemolytic str
staphylococci (resistant) from micrococci (susceptible).
• Principle: • Principle:
• Principle:
o Esculin hydrolysis produces esculetin, which reacts o Bile salts lyse pneumo
o Bacitracin inhibits cell wall synthesis; susceptibility with ferric ions to form a dark precipitate. presence of an autoly
indicated by a zone of inhibition.
• Method: bile esculin agar slant. • Method: BAP, sodium deoxych
• Method: BAP, Bacitracin Disk
• Expected Results: • Expected Results:
• Expected Results:
o Positive: Blackening of the medium. o Positive: Colony disin
o Positive: Zone of inhibition ≥10 mm (susceptible).
o Negative: No color change. o Negative: Intact colon
o Negative: No zone (resistant).
• Limitations: • Limitations:
• Limitations:
o Some organisms may not grow due to nutritional o Old cultures may yield
o Disk integrity affects performance. requirements.
• Quality Control:
• Quality Control: • Quality Control:
o Positive: Streptococcu
o Positive: Streptococcus pyogenes, Micrococcus o Positive: Enterococcus faecalis (black precipitate).
o Negative: Enterococcu
luteus.
o Negative: Escherichia coli, Streptococcus pyogenes. streptococci.
o Negative: Streptococcus agalactiae, Staphylococcus
aureus.
Source: Bailey’s and Scotts Diagnostic Microbiology (15th Edition)
1. Acetamide Utilization 2. Acetate Utilization 3. l-Alanine-7-amido-4-methylcourmar
• Purpose: Differentiate organisms based on the ability to use • Purpose: Differentiate organisms based on the ability to use • Purpose: Distinguish aerobic g
acetamide as the sole carbon source. acetate as the sole carbon source. coccobacilli that may appear g
• Principle: • Principle: • Principle:
o Bacteria producing acylamidase deaminate o Utilization of sodium acetate leads to alkaline pH, o Detects aminopeptida
acetamide, releasing ammonia.
turning the medium blue. negative organisms pr
o Ammonia increases pH, changing the medium from light.
green to royal blue. • Expected Results:
• Expected Results:
• Expected Results: o Positive: Blue color due to alkaline reaction.
o Positive: Blue fluoresc
o Positive: Blue color indicates acetamide utilization. o Negative: No color change; medium remains green.
o Negative: No fluoresc
o Negative: No color change; medium remains green. • Limitations:
• Limitations:
• Limitations: o Some E. coli strains may give false negatives.
o May not work with ob
o Heavy inoculum may lead to false negatives. • Quality Control:
• Quality Control:
• Quality Control: o Positive: Escherichia coli (growth; blue).
o Positive: Escherichia c
o Positive: Pseudomonas aeruginosa (growth; blue o Negative: Shigella sonnei (little growth; green).
color). o Negative: Staphylococ
o Negative: Escherichia coli (no growth; green color).
Source: Bailey’s and Scotts Diagnostic Microbiology (15th Edition)
, 4. Bacitracin Susceptibility Test 5. Bile Esculin Test 6. Bile Solubility Test
• Purpose: Differentiate β-hemolytic group A streptococci • Purpose: Identify enterococci and group D streptococci by • Purpose: Differentiate Streptoc
(susceptible) from other streptococci and distinguish their ability to hydrolyze esculin in the presence of bile. from other alpha-hemolytic str
staphylococci (resistant) from micrococci (susceptible).
• Principle: • Principle:
• Principle:
o Esculin hydrolysis produces esculetin, which reacts o Bile salts lyse pneumo
o Bacitracin inhibits cell wall synthesis; susceptibility with ferric ions to form a dark precipitate. presence of an autoly
indicated by a zone of inhibition.
• Method: bile esculin agar slant. • Method: BAP, sodium deoxych
• Method: BAP, Bacitracin Disk
• Expected Results: • Expected Results:
• Expected Results:
o Positive: Blackening of the medium. o Positive: Colony disin
o Positive: Zone of inhibition ≥10 mm (susceptible).
o Negative: No color change. o Negative: Intact colon
o Negative: No zone (resistant).
• Limitations: • Limitations:
• Limitations:
o Some organisms may not grow due to nutritional o Old cultures may yield
o Disk integrity affects performance. requirements.
• Quality Control:
• Quality Control: • Quality Control:
o Positive: Streptococcu
o Positive: Streptococcus pyogenes, Micrococcus o Positive: Enterococcus faecalis (black precipitate).
o Negative: Enterococcu
luteus.
o Negative: Escherichia coli, Streptococcus pyogenes. streptococci.
o Negative: Streptococcus agalactiae, Staphylococcus
aureus.
Source: Bailey’s and Scotts Diagnostic Microbiology (15th Edition)
